Genetically engineered strain of Saccharomyces cerevisiae, method for constructing the same and its use for brewing

Abstract

The present disclosure belongs to the field of bioengineering, and relates to breeding of industrial microorganisms, in particular to a genetically engineered strain of Saccharomyces cerevisiae , method for constructing the same, and its use for brewing, the genetically engineered strain of Saccharomyces cerevisiae heterogeneously overexpresses an acetaldehyde dehydrogenase gene ALD6, an acetyl-CoA synthase gene ACS1 and an alcohol acyltransferase gene AeAT9. The Saccharomyces cerevisiae strain with high yield of ethyl acetate and low yield of higher alcohols provided by the present disclosure not only maintains excellent ethanol fermentation characteristics, but also reducing the production of higher alcohols which adversely affect the comfort after drinking, which is of great significance for a well-maintained and strengthened flavor characteristics of Chinese Baijiu, an improved and stabilized quality thereof, and even a reform in the fermentation process thereof.

What is claimed is: 
     
       1. A genetically engineered strain of  Saccharomyces cerevisiae , wherein, the engineered strain heterologous overexpresses an acetaldehyde dehydrogenase gene ALD6, an acetyl-CoA synthase gene ACS1 and an alcohol acyltransferase gene AeAT9; the engineered strain further heterogeneously overexpresses an alcohol dehydrogenase gene ADH2; and the engineered strain does not express a porin gene POR2 responsible for transporting cytosolic pyruvate into the mitochondria. 
     
     
       2. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 1 , wherein, the acetaldehyde dehydrogenase gene ALD6 has the nucleotide sequence as shown in SEQ ID NO: 1; and/or the acetyl-CoA synthase gene ACS1 has the nucleotide sequence as shown in SEQ ID NO:2; and/or the alcohol dehydrogenase gene ADH2 has the nucleotide sequence as shown in SEQ ID NO:3; and/or the alcohol acyltransferase gene AeAT9 has the nucleotide sequence as shown in SEQ ID NO:4; and/or the porin gene POR2 responsible for transporting cytosolic pyruvate into the mitochondria has the nucleotide sequence as shown in SEQ ID NO:5. 
     
     
       3. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 1 , wherein, the acetaldehyde dehydrogenase gene ALD6 is connected to a strong promoter PGK1 P  (SEQ ID NO:7) and a terminator GIC1 T  (SEQ ID NO:8); and/or
 the acetyl-CoA synthetase gene ACS1 is connected to a strong promoter TEF1 P  (SEQ ID NO:9) and a terminator PGK1 T  (SEQ ID NO:10); and/or 
 the alcohol acyltransferase gene AeAT9 is connected to a strong promoter PGK1 P  (SEQ ID NO:7) and a terminator PGK1 T  (SEQ ID NO:10); and/or 
 the alcohol dehydrogenase gene ADH2 is connected to an inducible promoter HTX7 P  (SEQ ID NO:11) and a terminator PGK1 T  (SEQ ID NO:10). 
 
     
     
       4. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 2 , wherein, the acetaldehyde dehydrogenase gene ALD6 is connected to a strong promoter PGK1 P  (SEQ ID NO:7) and a terminator GIC1 T  (SEQ ID NO:8); and/or
 the acetyl-CoA synthetase gene ACS1 is connected to a strong promoter TEF1 P  (SEQ ID NO:9) and a terminator PGK1 T  (SEQ ID NO:10); and/or 
 the alcohol acyltransferase gene AeAT9 is connected to a strong promoter PGK1 P  (SEQ ID NO:7) and a terminator PGK1 T  (SEQ ID NO:10); and/or 
 the alcohol dehydrogenase gene ADH2 is connected to an inducible promoter HTX7 P  (SEQ ID NO:11) and a terminator PGK1 T  (SEQ ID NO:10). 
 
     
     
       5. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 1 , wherein, an original strain of the engineered strain is  Saccharomyces cerevisiae  CICC32315. 
     
     
       6. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 2 , wherein, an original strain of the engineered strain is  Saccharomyces cerevisiae  CICC32315. 
     
     
       7. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 3 , wherein, an original strain of the engineered strain is  Saccharomyces cerevisiae  CICC32315. 
     
     
       8. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 4 , wherein, an original strain of the engineered strain is  Saccharomyces cerevisiae  CICC32315. 
     
     
       9. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 5 , wherein, the acetyl-CoA synthetase gene ACS1, the aldehyde dehydrogenase gene ALD6 and the alcohol acyltransferase gene AeAT9 are sequentially connected, inserted into and replace a coding gene Gal80 region of a galactose transcription regulator in the  Saccharomyces cerevisiae ; the coding gene Gal80 has the nucleotide sequence as shown in SEQ ID NO:6. 
     
     
       10. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 6 , wherein, the acetyl-CoA synthetase gene ACS1, the aldehyde dehydrogenase gene ALD6 and the alcohol acyltransferase gene AeAT9 are sequentially connected, inserted into and replace a coding gene Gal80 region of a galactose transcription regulator in the  Saccharomyces cerevisiae ; the coding gene Gal80 has the nucleotide sequence as shown in SEQ ID NO:6. 
     
     
       11. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 7 , wherein, the acetyl-CoA synthetase gene ACS1, the aldehyde dehydrogenase gene ALD6 and the alcohol acyltransferase gene AeAT9 are sequentially connected, inserted into and replace a coding gene Gal80 region of a galactose transcription regulator in the  Saccharomyces cerevisiae : the coding gene Gal80 has the nucleotide sequence as shown in SEQ ID NO:6. 
     
     
       12. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 8 , wherein, the acetyl-CoA synthetase gene ACS1, the aldehyde dehydrogenase gene ALD6 and the alcohol acyltransferase gene AeAT9 are sequentially connected, inserted into and replace a coding gene Gal80 region of a galactose transcription regulator in the  Saccharomyces cerevisiae ; the coding gene Gal80 has the nucleotide sequence as shown in SEQ ID NO:6. 
     
     
       13. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 5 , wherein, the alcohol dehydrogenase gene ADH2 is inserted at a site of, and replaces an isoamyl acetate hydrogenase gene IAH1; the isoamyl acetate hydrogenase gene IAH1 has the nucleotide sequence as shown in SEQ ID NO:12. 
     
     
       14. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 6 , wherein, the alcohol dehydrogenase gene ADH2 is inserted at a site of, and replaces an isoamyl acetate hydrogenase gene IAH1; the isoamyl acetate hydrogenase gene IAH1 has the nucleotide sequence as shown in SEQ ID NO:12. 
     
     
       15. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 7 , wherein, the alcohol dehydrogenase gene ADH2 is inserted at a site of, and replaces an isoamyl acetate hydrogenase gene IAH1; the isoamyl acetate hydrogenase gene IAH1 has the nucleotide sequence as shown in SEQ ID NO:12. 
     
     
       16. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 8 , wherein, the alcohol dehydrogenase gene ADH2 is inserted at a site of, and replaces an isoamyl acetate hydrogenase gene IAH1; the isoamyl acetate hydrogenase gene IAH1 has the nucleotide sequence as shown in SEQ ID NO:12. 
     
     
       17. The genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 1 , wherein, the porin gene POR2 responsible for transporting cytosolic pyruvate into the mitochondria is knocked out. 
     
     
       18. A method for constructing the genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 1 , comprising: introducing into  Saccharomyces cerevisiae  the aldehyde dehydrogenase gene ALD6, the acetyl-CoA synthase gene ACS1, the alcohol acyltransferase gene AeAT9 and the alcohol dehydrogenase gene ADH2; and inactivating or knocking out the porin gene POR2 responsible for transporting cytosolic pyruvate into the mitochondria. 
     
     
       19. The method according to  claim 18 , further comprising:
 (1) obtaining a first recombinant strain through introducing the acetyl-CoA synthetase gene ACS1, the aldehyde dehydrogenase gene ALD6 and the alcohol acyltransferase gene AeAT9 into the  Saccharomyces cerevisiae , and replacing the coding gene Gal80 of the transcriptional regulator of galactose in  Saccharomyces cerevisiae  by homologous recombination; 
 (2) obtaining a second recombinant strain through introducing the alcohol dehydrogenase gene ADH2 into the first recombinant strain, and replacing the isoamyl acetate hydrogenase gene IAH1 in  Saccharomyces cerevisiae  by homologous recombination; and 
 (3) knocking out the porin gene POR2 responsible for transporting cytosolic pyruvate into the mitochondria in the second recombinant strain to obtain the genetically engineered strain of  Saccharomyces cerevisiae ; the knockout of the porin gene POR2 is realized by homologous recombination of POR2 and a KanMX resistance gene. 
 
     
     
       20. A method for brewing by fermentation, comprising the step of culturing the genetically engineered strain of  Saccharomyces cerevisiae  according to  claim 1  in a liquid medium.